Methods and compositions for culturing keratinocytes

Abstract

Methods, compositions, and kits are disclosed for culturing keratinocytes and for the treatment of wounds and burns, as well as cosmetic compositions. The methods and compositions include the use of conditioned medium from the culture of keratinocytes, where the medium may be used to further culture keratinocytes or used in compositions such as keratinocyte culture medium and pharmaceutical and cosmetic compositions.

1 . A method of culturing keratinocytes comprising contacting growing keratinocytes with a culture medium comprising conditioned medium, wherein said conditioned medium has previously been used to culture fetal keratinocytes.  
   
   
       2 . The method of  claim 1  further comprising contacting the growing keratinocytes with a cyclic adenosine monophosphate (cAMP)-elevating agent.  
   
   
       3 . The method of  claim 2  wherein the cAMP-elevating agent is selected from the group consisting of forskolin, cholera toxin, dibutyryl cAMP, isobutylmethylxanthine, theophylline, isoproterenol, and PGE2.  
   
   
       4 . The method of  claim 3  wherein the cAMP-elevating agent is forskolin.  
   
   
       5 . The method of  claim 4  wherein the forskolin is present at a concentration of about 0.8 ug/ml to about 5 ug/ml.  
   
   
       6 . The method of  claim 1  wherein the growing keratinocytes are fetal keratinocytes.  
   
   
       7 . The method of  claim 6  wherein the growing fetal keratinocytes and the fetal keratinocytes used to produce the conditioned medium are genetically identical.  
   
   
       8 . The method of  claim 1  wherein the culture medium comprises about 30-50% conditioned medium.  
   
   
       9 . The method of  claim 1  wherein the culture medium is produced by the steps of: 
 (a) culturing fetal keratinocytes in a keratinocyte culture medium for at least about eight hours to produce a conditioned medium; and    (b) mixing the conditioned medium with fresh keratinocyte culture medium so that the conditioned medium comprises about 30-50% by volume of the mixture.    
   
   
       10 . The method of  claim 9  further comprising adding a cAMP-elevating agent to the culture medium.  
   
   
       11 . The method of  claim 10  wherein step (b) is repeated about every day.  
   
   
       12 . The method of  claim 11 , wherein step (b) is repeated until a cell culture is produced wherein the cells of the cell culture comprise greater than about 99% keratinocytes.  
   
   
       13 . The method of  claim 11 , wherein step (b) is repeated until a cell culture is produced wherein the cells of the cell culture comprise greater than about 99.9% keratinocytes.  
   
   
       14 . The method of  claim 1  further comprising passing the keratinocytes when they are about 60-70% confluent.  
   
   
       15 . The method of  claim 1  wherein the fetal keratinocytes are grown in serum-free medium.  
   
   
       16 . A method of culturing keratinocytes comprising contacting growing keratinocytes with a culture medium comprising conditioned medium, wherein said conditioned medium has previously been used to culture keratinocytes under animal product-free conditions at all times.  
   
   
       17 . The method of  claim 16  further comprising contacting the growing keratinocytes with a cAMP-elevating agent.  
   
   
       18 . A composition for culturing keratinocytes comprising keratinocyte culture medium and conditioned medium in which fetal keratinocytes have been cultured.  
   
   
       19 . The composition of  claim 18  further comprising a cAMP-elevating agent.  
   
   
       20 . The composition of  claim 19  wherein the cAMP-elevating agent is selected from the group consisting of forskolin, cholera toxin, dibutyryl cAMP, isobutylmethylxanthine, theophylline, isoproterenol, and PGE2.  
   
   
       21 . The composition of  claim 20  wherein the cAMP-elevating agent is forskolin.  
   
   
       22 . The composition of  claim 21  wherein the forskolin is present at a concentration of about 0.8 ug/ml to about 5 ug/ml.  
   
   
       23 . The composition of  claim 18  wherein the conditioned medium comprises about 30-50% of the composition by volume.  
   
   
       24 . A culture medium produced by mixing fresh culture medium with a protein produced by the process of culturing fetal keratinocytes.  
   
   
       25 . The composition of  claim 24  further comprising a cAMP-elevating agent.  
   
   
       26 . A cell culture medium comprising conditioned medium that has previously been used to culture keratinocytes under conditions wherein said cultured keratinocytes are cultured at all times under animal product-free conditions.  
   
   
       27 . The composition of  claim 26  further comprising a cAMP-elevating agent.  
   
   
       28 . A keratinocyte culture that persists for more than about one year, wherein the cells of the culture comprise at least about 99% keratinocytes.  
   
   
       29 . A composition for promoting wound healing comprising a protein that is produced by growing fetal keratinocytes in culture.  
   
   
       30 . A composition for promoting wound healing comprising a protein that is produced by growing keratinocytes in culture, wherein the keratinocytes are grown by a method comprising contacting growing keratinocytes with a culture medium comprising conditioned medium, wherein said conditioned medium has previously been used to culture keratinocytes under conditions wherein said cultured keratinocytes are cultured at all times under animal product-free conditions.  
   
   
       31 . A method of modulating wound healing comprising contacting a wound with a protein from a conditioned medium produced by fetal keratinocyte culture.  
   
   
       32 . The method of  claim 31  further comprising contacting the wound with a cAMP-elevating agent.  
   
   
       33 . The method of  claim 32  wherein the cAMP-elevating agent is forskolin.